Neotropical Ichthyology, 14(3): e160024, 2016 Journal homepage: www.scielo.br/ni DOI: 10.1590/1982-0224-20160024 Published online: 30 September 2016 (ISSN 1982-0224)

First record of a male of hermaphroditus Costa, 2011 (: Cynolebiidae)

Waldir M. Berbel-Filho1,2, Helder M. V. Espírito-Santo3 and Sergio M. Q. Lima1

During an ichthyological survey in September 2015 at the Ceará-Mirim River estuary, Rio Grande do Norte State, northeastern Brazil, we collected a male of Kryptolebias hermaphroditus, a cynolebiid species that had been previously described as containing exclusively self-fertilizing . This is the first record of a male in this species, over 140 years after the discovery of the mangrove rivulid species from Brazil. Our discovery reinforces the need for more studies in K. hermaphroditus, as well as the potential of this species as a model for evolutionary studies due to its unique mating system. Durante uma amostragem ictiológica em setembro de 2015 no estuário do rio Ceará-Mirim, Estado do Rio Grande do Norte, Nordeste do Brasil, nós coletamos um macho de Kryptolebias hermaphroditus, uma espécie de rivulídeo que foi descrita como contendo apenas hermafroditas auto-fertilizantes. Este é o primeiro registro de um macho dessa espécie, mais de 140 anos depois da descoberta das espécies de rivulídeos de manguezais do Brasil. Nossa descoberta reforça a necessidade de mais estudos em K. hermaphroditus, assim como, o potencial desta espécie como um modelo para estudos evolutivos devido ao seu sistema reprodutivo único. Keywords: Hermaphroditism, Mangrove killifish, Mid-Northeastern Caatinga ecoregion, Mixed-mating system, Sexual dimorphism.

Introduction 2009; Costa et al., 2010). Because of its reproductive mixed- mated system, K. marmoratus has been a popular model Although the overall of Kryptolebias Costa, species for embryological, physiological, behavioral and 2004 is confusing (Costa, 2006), the K. marmoratus species genetic studies (Avise & Tatarenkov, 2015). While males group is well supported by both molecular (Tartarenkov et are rare in K. marmoratus, they are considered to be absent al., 2009) and morphological data (Costa et al., 2010). The in K. hermaphroditus (Costa, 2011; Tatarenkov et al., 2011), species comprising this group, K. marmoratus (Poey, 1880), despite this species having been known at least since 1868 K. ocellatus (Hensel, 1868) and K. hermaphroditus Costa, (previously known as K. ocellatus) and numerous museum 2011, inhabit estuarine areas associated with mangroves, and unpreserved specimens have been examined (Costa, ranging from southeastern United States of America to 2011). Due to the absence of males in fish collections, and southern Brazil (Costa, 2011). Besides this unusual brackish following a re-examination of the holotype of K. ocellatus, habitat, these species are also unique among aplocheiloid the monomorphic species from Brazil was killifishes due to the absence of females, which are replaced described as K. hermaphroditus (Costa, 2011). by hermaphrodites (Costa et al., 2010). Hermaphrodites of both species (K. marmoratus and Indeed, self-fertilizing hermaphroditism is the dominant K. hermaphroditus) typically have a black ocellus on the mode of reproduction in Kryptolebias marmoratus caudal peduncle and a dark-grey body side (Soto & Noakes, distributed from Florida to Venezuela (Tatarenkov et al., 1994; Costa, 2011). In K. marmoratus, males exhibit a bright 2011) and K. hermaphroditus occurring in the Atlantic orange coloration on the body flank and fins, an absent or Forest mangroves of Brazil (Lira et al., 2015). This poorly-defined ocellus on the caudal peduncle, and black clade represents the only occurrence of self-fertilizing margins on the caudal fins (Davis et al., 1990; Soto & hermaphroditism among (Tartarenkov et al., Noakes, 1994; Costa, 2011).

1Laboratório de Ictiologia Sistemática e Evolutiva, Departamento de Botânica e Zoologia, Universidade Federal do Rio Grande do Norte, Av. Senador Salgado Filho 3000, 56078-970 Natal, RN, Brazil. (SMQL) [email protected], (WMBF) [email protected] (corresponding author) 2Department of BioSciences, College of Science, Swansea University, SA2 8PP, Swansea, Wales, United Kingdom 3Post-Doctoral Program, Instituto Nacional de Pesquisas da Amazônia, Av. André Araújo, 2936, Petrópolis, 69067-375 Manaus, AM, Brazil. [email protected] 1 Neotropical Ichthyology, 14(3): e160024, 2016 2 A male Kryptolebias hermaphroditus

In a recent collection in the Ceará-Mirim River estuary, Results we found a Kryptolebias hermaphroditus individual with a male phenotype. Considering that this species has been All specimens were identified as Kryptolebias described as being completely composed of hermaphrodites, hermaphroditus on the basis of having 1-4 teeth in the the main objective of the present study is to record and vomer and a color pattern with silvery or pale golden describe the first occurrence of a male K. hermaphroditus. spots on the dorsal part of the flank. The male individual had bright orange coloration on the body flank and fins Material and Methods (mainly dorsal, anal and caudal), and black margins on the anal and caudal fins (Fig. 1), as in males of K. In September 2015, during a nocturnal exploratory marmoratus. fieldwork being conducted with the objective of designing Comparing male and hermaphroditic phenotypes an ecological study on Kryptolebias hermaphroditus at the of the captured specimens, both have a dark ocellus on Ceará-Mirim River estuary (05°40’25.9”S 35°14’14.5”W, the caudal peduncle; however, the ocellus is faint on the see Lira et al., 2015 for a map and detailed description of the male specimen. The hermaphrodites consistently exhibit area), we observed a bright orange individual of Kryptolebias a brown or grey flank coloration, as well as hyaline (Fig. 1). This specimen together with specimens exhibiting unpaired fins with pale grey dots on the basal portion, a more typical hermaphroditic appearance, were captured whereas the male individual exhibits unpaired fins with by hand and with the help of a hand sieve (50 x 60 cm, 1 orange coloration (Fig. 2). mm mesh), under permit #30532-1/2011 issued by ICMBio/ SISBIO. The fishes were euthanized using clove oil, Description. Morphometric data of male (30.1 mm SL) fixed in formalin solution, transferred to an 70% alcohol in Table 1. Urogenital papilla cylindrical in male, pocket- solution and then deposited in the ichthyological collection like shape in hermaphrodites (Fig. 3). Dorsal fin rounded. of Universidade Federal do Rio Grande do Norte (UFRN Anal fin sub-trapezoidal, distal margin slightly damaged; 3774, 8 ex.). Some other individuals, as well as a small anal-fin rays 4 and 5 longer than other anal-fin rays. Caudal fin-clip (TIUFRN 3481; tissue collection of UFRN), were fin oval, deeper than long. Pectoral fin short and rounded. directly stored in 96% ethanol for molecular analysis. Pelvic fin elliptical. Pelvic-fin bases medially separated by Measurements and counts for the orange individual short interspace. Dorsal-fin origin at vertical between 9th follow Costa (1995). Kryptolebias species identification and 10th anal-fin rays. Dorsal-fin rays 8; anal-fin rays 11; was carried out using the key proposed by Costa (2009), and caudal-fin rays 30; pectoral-fin rays 13; pelvic-fin rays 6. later confirmed using Costa (2011). To identify male traits, Scales small, cycloid. Frontal squamation E-patterned. we used the information described for the closely related Longitudinal series of scales 47; transverse series of scales species K. marmoratus (Davis et al., 1990; Soto & Noakes, 13; scale rows around caudal peduncle 25. Contact organs 1994; Costa, 2011). absent.

Fig. 1. Kryptolebias hermaphroditus, male, UFRN 3774, 30.1 mm SL (three days after sampling); Ceará-Mirim River estuary, Extremoz, Rio Grande do Norte, Brazil. Neotropical Ichthyology, 14(3): e160024, 2016 W. M. Berbel-Filho, H. M. V. Espírito-Santo & S. M. Q. Lima 3

Fig. 2. Kryptolebias hermaphroditus, male (below) 30.1 mm SL, hermaphrodite (above) 35.5 mm SL, UFRN 3774, Ceará- Mirim River estuary, Extremoz, Rio Grande do Norte, Brazil.

Table 1. Morphometric data of the Kryptolebias hermaphroditus, male (UFRN 3774). Standard length (mm) 30.16 Percents of standard length Body depth 17.11 Caudal-peduncle depth 13.23 Predorsal length 73.18 Prepelvic length 62.10 Length of dorsal-fin base 12.33 Length of anal-fin base 15.78 Caudal-fin length 25.86 Pectoral-fin length 17.90 Pelvic-fin length 6.63 Head length 25.10 Percents of head length Head depth 59.31 Head width 71.07 Fig. 3. Urogenital papillae of Kryptolebias hermaphroditus Snout length 29.33 in ventral view; UFRN 3374: a, cylindrical shape in male, Lower-jaw length 23.51 30.1 mm SL; b, pocket shape in hermaphrodite, 35.5 mm Eye diameter 26.55 SL. Scale bars = 1 mm. Neotropical Ichthyology, 14(3): e160024, 2016 4 A male Kryptolebias hermaphroditus

Coloration. Flank dark grey on anterior and dorsal specimens with light grey and dark orange spots on the portion of head, from greyish to orange on posterior and flanks, grey humeral and caudal peduncle spots and dark ventral portions. Side of the body intensely pigmented grey to black distal zones of unpaired fins. However, this with faint dark grey stripe between postorbital region author recognized that this material was not preserved and caudal-fin base, and small irregular somewhat for study in any scientific collection and needed posterior zigzag pattern composed of small orange spots more confirmation. Posteriorly, Costa (2011) described K. concentrated on anterior half of body side. Inconspicuous hermaphroditus as only composed by hermaphrodites, dark grey humeral blotch; rounded dark spot with faint discarding the relevance of the two previous unconfirmed yellowish grey margin on dorsal end of caudal peduncle. reports. In both cases male phenotypes were reported for Rio Side of head light brown on dorsal portion, pale golden de Janeiro, where K. ocellatus (males and hermaphrodites, with dark grey dots ventrally. Jaws grey. Iris brown, with previously known as K. caudomarginatus Seegers) occurs narrow pale yellow margin around pupil. Dorsal, anal and in syntopy with K. hermaphroditus making these records caudal fins hyaline with dark orangish-brown on basal more doubtful. The potential records described above, the and medial portions of fins; black stripe on distal anterior presence of males on populations of K. marmoratus (Avise margin of anal and lower portion of caudal fin. Pectoral & Tatarenkov, 2015), as well as the cryptic and complex and pelvic fins hyaline. Bright orange coloration faded mangrove microhabitats inhabited by K. hermaphroditus, a little after sampling, probably due to stress and non- may have led Costa (2006) and Lira et al. (2015) to not fully natural conditions (Figs. 1-2). discard the presence of males of K. hermaphroditus, despite extensive sampling and no reliable scientific evidence of Ecological notes. The sampling locality had clear water males have been made available up to that moment. and the fishes could be observed using flashlights. The The recently collected specimen of Kryptolebias Kryptolebias hermaphroditus male was captured in an hermaphroditus with male appearance, described herein, ephemeral pool at higher elevations among the estuary’s matches the description of the putative K. hermaphroditus mangroves. This pool had at least four fiddler crabs Uca( males mentioned by Costa (2006) and K. marmoratus maracoani Latreille, 1802) burrow openings and, prior males (Davis et al., 1990; Soto & Noakes, 1994; Costa, to our approach, the male fish was resting on the water’s 2011). Although gonadal histological evidence was not surface near one of these burrows. During our sampling surveyed in order to maintain the unique specimen attempts, the fish repeatedly hid and re-appeared among integrity, its morphological characters support this the crab burrows, appearing periodically in different first documentation of a male K. hermaphroditus. This openings until it was caught. Time between reappearances evidence includes orange body and fins, faded ocellus, was about one minute. black margins of anal and caudal fins and cylindrical Large (23.2-35.5 mm SL) hermaphrodites were also urogenital papillae (Costa, 2011). Once the color pattern of collected in the same area as the male, in shallow pools males is the most conspicuous character for differentiating formed in the edge of the mangroves; in the inner and cynolebiid species (Costa, 2003), the discovery of a K. deeper pools, Poecilia vivipara Bloch & Schneider, hermaphroditus male may reveal important morphological 1801 was the dominant species. Guavina guavina differences between this species and K. marmoratus. (Valenciennes, 1837), potential predator of Kryptolebias Regardless the presence of an ocellus on the caudal hermaphroditus, were also active during the night and peduncle, it appeared faded in comparison to that of observed entering the same large crab burrows. Salinity hermaphrodite individuals. According to Soto & Noakes in these pools, previously recorded in January 2015 at the (1994), the most distinctive characters attributed to same site, was 34-36 ppm during the low tide. Kryptolebias marmoratus males are: a bright orange overall coloration and the absence of a distinct caudal Discussion ocellus. However, they also considered that individuals with intermediate phenotypes (with orange color and a Over the 140 years, since the description of Kryptolebias faded ocellus) could also be males. Davis et al. (1990) ocellatus (as was long called hermaphroditic species who had previously mentioned that it is not possible yet to was later described as K. hermaphroditus) from a mangrove distinguish primary and secondary males, the latter of in Rio the Janeiro, no males of this hermaphroditic species which result from sex change of functional hermaphrodites from the Brazilian coast have ever been found (Costa, into males due to environmental conditions (Harrington, 2011). However, potential miswritten on previous literature 1971). Huber (1992) cited the presence of ocellus in (Costa, 2006; Lira et al., 2015) needs to be clarified for a secondary males of K. ocellatus, which agrees with our better understanding of the issue. Both studies used the specimen description. However, as discussed above, this subjective term “rare” while discussing the existence of report was not afterwards confirmed. males in K. hermaphroditus. Costa (2006) had already Primary males of Kryptolebias marmoratus can be mentioned two reports of potential K. hermaphroditus readily produced in laboratories by inducing eggs to males based on pictures and brief descriptions of live temperatures lower than those usually found in mangrove Neotropical Ichthyology, 14(3): e160024, 2016 W. M. Berbel-Filho, H. M. V. Espírito-Santo & S. M. Q. Lima 5 areas (<20°C), while secondary males can be induced by (Costa, 2006, 2011), evolutionary history (Costa et al., 2010; maintaining individuals at higher temperatures (>30°C), Tatarenkov et al., 2009, 2011), and geographic distribution which are similar to those found on populations with higher (Sarmento-Soares et al., 2014; Lira et al., 2015), there is rate of males (Turner et al., 2006; Earley et al., 2012). Their little available information regarding natural history, induction in the lab at naturally occurring temperatures ecology and population dynamics. Despite the large gap suggests that, in the wild, secondary males may be the in our knowledge of the biology of K. hermaphroditus, most ecologically relevant type of male (Turner et al., the recent studies above mentioned, including the present 2006). This may also be the case for K. hermaphroditus one, suggest some interesting perspectives for studies on in northeastern Brazil, where higher temperatures are evolution, morphology, behavior and physiology across the predominant due to the Equator’s line proximity. species’ wide geographic distribution in the mangroves of Davis et al. (1990) demonstrated distinct male the Brazilian coast. phenotypes within Kryptolebias marmoratus. One, from Twin Cays, , exhibited a dark orange color pattern, Acknowledgements an inconspicuous caudal ocellus, and caudal black margins along the dorsal, anal and caudal fins. The other one, from We thank Lucas Medeiros, Mateus Lira and Roney Florida, USA, exhibited a bright orange coloration almost Paiva for helping with fieldwork, and to Luciano Barros- devoid of dark chromatophores, except the head. Ellison et Neto for providing the pictures of the live specimens. al. (2012) also found a male of K. marmoratus with orange Carlos Eduardo Alencar provided information about coloration, faded ocellus and black borders on the anal and mangrove crabs in the Ceará-Mirim estuary. Graco caudal fins in Belize; a pattern similar to that of the K. Viana and Centro de Treinamento em Aquicultura da hermaphroditus male herein described. Universidade Federal do Rio Grande do Norte provided Between 2011 and 2015, during extensive surveys at the logistical support. Waldir Berbel-Filho receives a PhD Ceará-Mirim River estuary (Lira et al., 2015), we collected scholarship from Science without Borders Program/CNPq 107 hermaphrodites before finding this single male. (process #233161/2014-7) and Helder Espírito-Santo This apparent rarity of males aligns with the extremely received a Postdoctoral fellowship from PDJ Program/ low degree of heterozygosis reported for populations of CNPq (process #168228/2014-9). Kryptolebias hermaphroditus from southeastern Brazil (Tartarenkov et al., 2011). Together with absence of male References specimens in all known populations (Costa, 2011; Lira et al., 2015), these findings suggest that self-fertilizing Avise, J. C. & A. Tatarenkov. 2015. Population genetics and hermaphroditism is the major mode of reproduction in K. evolution of the mangrove Kryptolebias marmoratus, hermaphroditus. However, the persistent occurrence of the world’s only self-fertilizing hermaphroditic . male individuals in K. marmoratus, but at low densities Journal of Fish Biology, 87: 519-538. (varying from 1% in Florida to 10-25% in Belize, Davis Costa, W. J. E. M. 1995. Pearl killifishes: the Cynolebiatinae: et al., 1990), suggests that sexual outcrossing serves systematics and biogeography of the Neotropical annual fish an adaptive role within natural populations, possibly subfamily. Neptune city, TFH Publications, 128p. contributing to genetic diversity that helps the species to Costa, W. J. E. M. 2003. Family . Pp. 526-548. In: Reis, deal with new environmental pressures, such as parasites R. E., S. O. Kullander & C. J. Ferraris Jr. (Eds.). Check list loads (Ellison et al., 2011, 2013). of freshwater fishes of South and Central America. Porto The occurrence of self-fertilization in Kryptolebias Alegre, Edipucrs. marmoratus has made that species a popular model for Costa, W. J. E. M. 2006. Redescription of Kryptolebias population genetics, development, evolutionary biology, ocellatus (Hensel) and K. caudomarginatus (Seegers) behavior, among others (for reviews see Earley et al., 2012; (Teleostei: Cyprinodontiformes: Rivulidae), two killifishes Taylor, 2012; Avise & Tatarenkov, 2015; Turko & Wright, from mangroves of south-eastern Brazil. Aqua Journal of 2015). Nonetheless, there have been relatively few studies Ichthyology and Aquatic Biology, 11: 5-12. on the South American lineage of K. hermaphroditus, Costa, W. J. E. M. 2009. Peixes aploqueilóideos da Mata possibly due to its misidentification as K. marmoratus Atlântica brasileira: história, diversidade e conservação. Rio (Seegers, 1984). Additionally, the difficulties of working de Janeiro, Museu Nacional. 171p. in mangrove environments, which represent a complex, Costa, W. J. E. M. 2011. Identity of Rivulus ocellatus and a multi-dimensional, and cryptic habitat with variable new name for a hermaphroditic species of Kryptolebias environmental and physical features could also contributed from southeastern Brazil (Cyprinodontiformes: Rivulidae). to the scarcity of studies on the South American lineage Ichthyological Exploration of Freshwaters, 22: 185-192. (Taylor, 2012). Costa, W. J. E. M., S. M. Q. Lima & R. Bartolette. 2010. The discovery of a male specimen encourages in Kryptolebias and the evolution of self- more studies on Kryptolebias hermaphroditus. While fertilizing hermaphroditism. Biological Journal of the researchers have addressed some aspects of systematics Linnean Society, 99: 344-349. Neotropical Ichthyology, 14(3): e160024, 2016 6 A male Kryptolebias hermaphroditus

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